We plan to exploit a Drosophila melanogaster RNA polymerase II mutant we recently isolated to elucidate functionally-importnat structural features of DNA-dependent RNA polymerase II, to begin genetic characterization of RNA polymerases from a multi-cellular eukaryote, and to study the biology of eukaryotic transcription. The Drosophila mutant (called AmaC4) is the first eukaryotic organism described which is resistant to the effects of the RNA polymerase inhibitor gamma-amanitin because it contains an altered, amanitin-resistant RNA polymerase II. We plan a detailed biochemical comparison of higly-purified mutant and wild-type polymerase II aimed at identifying the subunit altered by the mutation and at defining that subunit's role in RNA polymerase function. We also plan to refine our preliminary genetic localization of the AmaC4 mutation by performing a series of recombination and cytogenetic mapping experiments. Using the knowledge gained from the mapping experiments we will attempt to isolate additional RNA polymerase II mutants, especially conditional-lethals. Utilizing the RNA polymerase mutants we will investigate the regulation of biosynthesis of RNA polymerase II in vivo, and we will analyze the eukaryotic transcription apparatus at the biochemical, genetic, and biological levels.